This invention relates to the field of DNA recombinant technology. More specifically, this invention relates to fusion proteins comprising human IFN-alpha2b and human TM alpha1 and pharmaceutical compositions containing the fusion proteins and methods for using them.
Interferons (IFN) are a family of polypeptides synthesized and secreted by a large variety of eukaryotic cells in response to viral infections or to various synthetic and biological inducers, including viral constituents, double stranded RNA, and mitogens. Human IFNs are classified into two major groups. The IFNs-alpha, -beta, and -omega are designated type I IFNs, and IFN-gamma is designated type II IFN. All type I IFNs exhibit high homology in their primary, secondary, and tertiary structures. They interact with the same receptor and activate similar transcriptional signaling pathways, eliciting a similar range of biological responses, including antiviral, antiproliferative, and immunomodulatory activities. Binding to a quite distinct cell surface receptor than type I IFN, Type II IFN differs from type I IFN in many aspects, such as the structure and induction of the gene, IFN's antigenicity, and biological responses. Type II IFN has a different range of immune functions such as macrophage activation.
In the human IFN system, there is only one IFN-beta, one IFN-omega, and one IFN-gamma gene, and there are at least 14 nonallelic IFN-alpha genes, which are located on chromosome 9 (Allen and Fantes, Nature 287:408, 1980; Owerback et al., Proc. Natl. Acad. Sci. 78:3123, 1981; Henco et al., J. Mol. Biol. 185:227, 1985; Diaz et al., J. Interferon Res. 11, S85, 1991; Diaz et al., J. Interferon Res. 13:61, 1993). Among the IFN-alpha genes, IFN-alpha2 (also called IFN-alpha A) gene locus is also found to contain three allelic variants, IFN-alpha2a, IFN-alpha2b, and IFN-alpha2c (Goeddel et al., Nature 287:411, 1980; Streuli et al., Science 209:1343, 1980; Lee et al., J. Interferon Res. 15:341, 1995). These three variants are unique among IFN-alpha genes in coding for mature proteins of 165 amino acids, since all other IFN-alpha proteins have 166 amino acids. Analysis of the cloned DNA sequences of these three IFN-alpha2 variants indicated they differ from each other in nucleotides(nt) at one or two positions (nt 137 and 170) in the coding region of the gene, resulting in a substitution of a lysine for arginine at position 23 in the mature IFN-alpha2a protein and an arginine for histidine at position 34 in the mature IFN-alpha2c proteins. Differing in only a few nucleotides, the purified IFN-alpha2a, 2b, and 2c are shown to differ significantly in their biologic and antigenic properties, indicating that differences in the amino acid sequences at position 23 and 34 may be significant in changing the immunogenicity as well as the structure and function of IFN-alpha2 (Von Gabain et al., Eur. J Biochem. 190:257, 1990). Naturally, IFN-alpha2b is predominant in the IFN-alpha2 species, as the IFN-alpha2 is predominant in the IFN-alpha species produced by normal human leukocytes (Emanuel and Pestka, J. Biol. Chem. 268: 12565, 1993; Gewert et al., J. Interferon Res. 13: 227, 1993; Dopaola et al., J. Interferon Res. 14:325, 1994). Among more than 24 IFN-alpha species identified so far from gene and protein sequence data, the predominant subspecies, IFN-alpha 2, is the most intensively studied (Weissmann and Weber, Prog Nucleic Acid Res. Mol. Biol. 33:251, 1986; Zoon, K. C., Interferon 9:1, 1987; Pestka et al., Ann. Rev. Biochem. 56:727, 1987).
Human IFN-alpha2 was among the first of the IFNs to be cloned by recombinant DNA technology. The recombinant version of IFN-alpha2, such as IFN-alpha2a, alpha2b or alpha2c, consists of a single unglycosylated species of IFN protein with a molecular weight of 19 Kd and a pI in the range of 5.5-6.5. Two IFN-alpha2 recombinant products, IFN-alpha2a (ROFERON, Hoffman-La Roche) and IFN-alpha2b (INTRON, Schering Plough), are commercially available. They are approved worldwide for the treatment of a variety of diseases including various cancers, particularly hematological malignancies such as hairy cell leukemia and chronic myelogenous leukemia, and viral induced disorders, such as hepatitis (Main et al., Antivir. Chem. Chemother. 9:449, 1998; Oren et al., Ann. Hematol. 77:187, 1998; Bruno et al., Ann. Intern. Med. 128:956, 1998; Hassanein et al., J. Viral. Hepat. 3:333, 1996; Dorr, R. T., Drugs 45:177, 1993).
Many IFN-alpha hybrids, conjugates and chimeras are disclosed in attempt to create IFN-alpha molecules with advantageous properties (U.S. Pat. Nos. 4,678,751; 5,071,761; 5,738,846; 5,594,107; Sperber et al., Antiviral. Res. 22:121, 1993; Rasch et al., Dig. Dis. Sci. 43:1719, 1998; He et al., J. Cancer Res. Clin. Oncol. 125:77, 1999).
A standard cell-free protein extract preparation from the thymus gland, known as thymosin fraction V (TF5) (U.S. Pat. No. 4,082,737), was demonstrated to be a potent immunopotentiating preparation. TF 5 can suppress to various extents immune deficiency diseases and can also act in lieu of the thymus gland to reconstitute immune functions in thymic deprived and/or immunodeprived individuals (Wara et al., N. Engl. J. Med 292: 70, 1975). Analytical polyacrylamide gel electrophoresis and isoelectric focusing have demonstrated that TF5 consists of a number of polypeptides termed thymosin, with molecular weights ranging from 1,000 to 15,000.
The first of these peptides to be purified to homogeneity and sequenced from TF5 was called thymosin alpha 1 (TM-alpha1) (Goldstein et al., Proc. Natl. Acad. Sci. 74:725, 1977; U.S. Pat. No. 4,079,127). The chemical synthesis of TM-alpha1 by solution and solid phase synthesis techniques is described in U.S. Pat. Nos. 4,148,788 and 5,856,440. Identical to the native TM-alpha1 in the biological potent and amino acid sequence with lack of the N-terminal acetyl group, recombinant TM-alpha1 can be produced in E. coli by recombinant DNA cloning techniques (Wetzel et al., Biochemistry 19:6096, 1980). TM-alpha1 analogs and derivatives also have been produced, U.S. Pat. Nos. 4,116,951 and 5,512,656. TM-alpha1 is a 28 amino acid acidic peptide with a molecular weight of 3,100 and a pI in the range of 4.0-4.3. TM-alpha1 maintains many of the biologic effects of TF5 and has been found to be 10 to 1,000 times more active than TF5 in a number of bioassay systems designed to measure the maturation and function of T lymphocytes.
TM-alpha1 potentiates the immune system by stimulating alpha- and gamma-interferon production, increasing T cell numbers, increasing production of macrophage migration inhibitory factor, inducing expression of T-cell markers, including interleukin-2 receptors, and improving T-cell helper cell activity (Marshall et al., J. Immunol. 126:741, 1981; Mutchnick et al., Clin. Immunol. Immunopathol. 23:626, 1982; Low et al., Thymus 6:27,1984; Sztein et al., Proc. Natl. Acad. Sci. 83:6107, 1986; Serrate et al., J. Immunol. 1939:2338,1987; Baxevanis et al., Immunopharm. 13:133, 1987; and, Svedersky, L. P., Eur. J. Immunol. 12:244, 1982). TM-alpha1 is currently under clinical trial to determine its efficacy in the treatment of immunodeficiency diseases, immunodepressed cancer patients and chronic active hepatitis (Goldstein, A. L., Cancer Invest. 12:545, 1994; Lopez et al., Ann. Oncol. 5:741, 1994; Garaci et al., Eur. J. Cancer. 31A:2403,1995; Garaci et al., Mech. Ageing. Dev. 96:103, 1997; Bonkovsky, H. L., Hepatology 26(3 Suppl 1):143S, 1997; Liaw, Y. F., J. Gastroenterol. Hepatol. 12:S346, 1997).
Clinical studies have demonstrated that the combination therapy of IFN-alpha2 and TM-alpha1 is more effective than either IFN-alpha2 and TM-alpha1 alone in treatment of cancers and chronic hepatitis (Garaci et al., Int. J. Clin. Lab. Res. 24:23, 1994; Garaci et al., J. Immunother. 13:7, 1993; Garaci et al., Eur. J. Cancer 31A:2403, 1995; U.S. Pat. No. 5,849,696; Rasi et al., Gut 39:679, 1996; Sherman et al., Hepatology 27:1128, 1998; Moscarella et al., Liver 18:366, 1998).
Although the exact mechanism of action of IFN-alpha2 and TM-alpha1 in the treatment of the above mentioned diseases is not fully understood, their biological activities are mediated by binding to specific cell surface receptors. IFN-alpha2 and TM-alpha1 each bind to their respective receptors, resulting in a biological signal transduction to various effector cells. Studies of the receptor binding for IFN-alpha2 and TM-alpha1 indicate that IFN-alpha2 and TM-alpha1 share a sequence homology and compete with each other for high-affinity receptors on murine thymocytes. These studies showed that binding of .sup.125I-labelled octapeptide (fragment 130-137 of IFN-alpha2) to high-affinity receptors on thymocytes is efficiently inhibited by both unlabelled IFN-alpha2 and unlabelled TM-alpha1 (Zav'yalov et la., FEBS Lett. 278:187, 1991). Further studies also showed that prothymosin alpha (proTM-alpha) competes with .sup.125I-labelled IFN-alpha2 for binding the same receptor on human fibroblasts (Zav'yalov et la., Mol. Biol. 32: 425, 1995). ProTM-alpha, a 113 amino acid thymic polypeptide, was named because it was thought to be a precursor to TM-alpha1. ProTM-alpha includes thymosin-alpha1 as its 28 N-terminal amino acids and possess the same approximate quantitative and qualitative biological activity that has been ascribed to TM-alpha1 (U.S. Pat. No. 4,716,148; Smith, M. R., Leukemia and Lymphoma 18:209, 1995). This direct competition between IFN-alpha2 and TM-alpha1/proTM-alpha for a single cell surface receptor indicates that a single receptor is capable of binding both IFN-alpha2 and TM-alpha1/proTM-alpha. However, it is not yet clear whether the heterogeneity in IFN-alpha2 and TM-alpha1/proTM-alpha binding is due to the existence of a shared receptor subunit within the multisubunit complexes of the IFN-alpha2 and TM-alpha1/proTM-alpha receptors, or due to the existence of a coreceptor which is distinct from that which binds IFN-alpha2 alone or TM-alpha1/proTM-alpha alone, or due to the distinct binding manner in ligand- receptor interactions that results in different tertiary structures within the intracellular portion of the receptor chains leading to a specific signaling (Lewerenz et al., J. Mol. Biol. 282:585, 1998; Russell-Harde et al., Biochem. Biophy. Res. Communic. 255:539, 1999). Therefore, the receptor, which binds to the IFN-alpha2/TM-alpha1 fusion proteins, will be referred to herein as the IFN-alpha2/TM-alpha1 receptor.